Enhanced mediated DNA transfer

ABSTRACT

A method to increase the efficiency of transduction of hematopoietic and other cells by retroviruses includes infecting the cells in the presence of fibronectin or fibornectin fragments. The fibronectin and fibronectin fragments significantly enhance retroviral-mediated gene transfer into the cells, particularly hematopoietic cells including committed progenitors and primitive hematopoietic stem cells. The invention also provides improved methods for somatic gene therapy capitalizing on enhanced gene transfer, hematopoietic cellular populations, and novel constructs for enhancing retroviral-mediated DNA transfer into cells and their use.

PRIORITY CLAIM

This application is a divisional of U.S. application Ser. No. 09/394,867filed Sep. 13, 1999, which is a continuation of U.S. application Ser.No. 08/536,891 filed Sep. 29, 1995, which is now U.S. Pat. No. 6,033,907issued Mar. 7, 2000, which is a continuation-in-part of PCT ApplicationNo. PCT/US95/03817 filed Mar. 27, 1995, which is a CIP of U.S. patentapplication Ser. No. 08/218,355 filed Mar. 25, 1994 which is now U.S.Pat. No. 5,686,278 issued Nov. 11, 1997, all of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates generally to methods for increasing theefficiency of transduction of cells by viruses, and more particularly tomethods for enhancing viral-mediated gene transfer into cells utilizingfibronectin and/or fibronectin fragments.

BACKGROUND OF THE INVENTION

Progress in understanding the molecular basis of many human diseases aswell as improvement in gene transfer technology has led to recentattempts to develop protocols for somatic gene therapy for severegenetic diseases. Currently, promising disease candidates for human genetherapy include those in which an enzyme or other protein is defectiveor missing, where the level of enzyme or protein does not need to beexactly regulated, especially those that are constitutively regulated,and those defects which are found in the patient's bone marrow.

For example, one disease candidate for gene therapy is adenosinedeaminase (ADA) deficiency which results in severe combinedimmunodeficiency disease (SCID). ADA deficient patients have little orno detectable enzyme in bone marrow cells. However, ADA deficiency hasbeen cured by matched bone marrow transplantation. ADA normal cells havea selective advantage over ADA deficient cells and will normallyrepopulate the patient's bone marrow.

Bone marrow cells are a good target for somatic gene therapy becausebone marrow tissue is easily manipulated in vitro and containsrepopulating cells. Alternatively, human cord blood has previously alsobeen demonstrated to contain a large number of primitive progenitorcells. Successful gene transfer into hematopoietic stem cells, the longterm repopulating cells, may lead to lifelong cures for a variety ofdiseases manifested in the progeny of these cells.

Gene transfer into, and long term gene expression in, repopulating stemcells has been achieved in murine models by a number of investigators.However, in vivo experiments in larger animals, such as dogs andprimates, have met with limited success, largely due to the lowefficiency of infection of primitive hematopoietic stem cells. Thelimitations of current gene transfer technology are further complicatedwhen applied to human protocols by several factors, including the lownumbers of stem cells present in adult bone marrow (ABM), the lack ofsuitable methods to purify these cells, and the low fraction of suchprimitive cells in cell cycle.

In both murine and large animal experiments involving bone marrow cells,it has been noted that the most successful protocols utilizecocultivation of target cells with retroviral producer cell lines. Also,most of the FDA-approved gene transfer trials in humans rely onrecombinant retroviral vectors for gene transduction. Recombinantretroviral vectors are desirable for gene therapy because theyefficiently transfer and precisely and stably integrate exogenous DNAinto cellular DNA. These vectors contain exogenous DNA for gene transferand are further modified to eliminate viral pathogenicity. Because ofthese modifications, viral production is generally accomplished usingretrovirus packaging cells. However, for clinical gene therapy,cell-free transduction is more desirable due to concerns aboutbio-safety and quality control. Unfortunately, efficient gene transferinto hematopoietic cells such as stem cells has generally not beenpossible without cocultivation with virus-producing cells.

Recently, it has been shown that gene transfer efficiency can beincreased by exposing target cells to stromal cells during infection.Stromal cells are a major component of the hematopoieticmicroenvironment (HM). The HM consists of an organized network ofmacrophages, stromal cells, endothelial cells, adipocytes and a complexextracellular matrix (ECM) made up of a variety of defined adhesionmolecules. ECM molecules such as laminin, collagen, thrombospondin,proteoglycans, glycosaminoglycans and fibronectin provide anchoragesites for both hematopoietic cells and growth factors. The mechanismunderlying this promoting effect of stroma on retroviral infection isunclear, but it has been known for some time that physiologic regulationof the proliferation and differentiation of hematopoietic cells occurswhen these cells are in direct contact with cells of the HM.

Efficient gene transfer into long term repopulating hematopoietic stemcells and other cells remains problematic, inhibiting the widespreadapplication of gene transfer protocols for curative therapy ofhematopoietic and other diseases at present. A need persists for methodsfor efficient transfer of genetic material into mammalian cells withoutthe dangers and limitations of past methods. The present inventionaddresses these needs.

SUMMARY OF THE INVENTION

Briefly, one preferred embodiment of this invention provides a methodfor increasing the frequency of transduction of hematopoietic cells by aretrovirus vector. The method includes infecting viable hematopoieticcells with a replication-defective recombinant retrovirus vector in thepresence of substantially pure fibronectin and/or fibronectin fragmentseffective to increase the frequency of cellular transduction by theretrovirus. The fibronectin and/or fibronectin fragments can be derivedfrom naturally-occurring materials or can be synthetically derived (e.g.genetically engineered by recombinant or chemical synthesis techniques),or derived from a combination of naturally-occuring and syntheticmaterials. In addition, it will be understood that the fibronectinpolypeptide or polypetides utilized in the invention may includemutations to the naturally-occurring fibronectin amino acid sequencewhich nonetheless provide functional polypeptides having the adhesionproperties necessary for achieving enhanced transduction in accordancewith the invention.

Another preferred embodiment of the invention provides a method forproducing transduced hematopoietic cells which includes infecting viablehematopoietic cells with a replication-defective recombinant retroviruscarrying exogenous DNA in the presence of immobilized fibronectin,immobilized fibronectin fragments, or an immobilized mixture thereof inamounts effective to increase the frequency of cellular transduction bythe retrovirus.

Another preferred embodiment of the invention provides an improvedmethod for cellular grafting. The method includes the steps of obtainingviable hematopoietic cells from an animal donor; infecting thehematopoietic cells with a recombinant retrovirus vector to producetransduced viable hematopoietic cells, the infecting being in thepresence of fibronectin and/or a fragment thereof in immobilized formand effective to increase the frequency of transduction; and introducingthe transduced viable hematopoietic cells into an animal recipient as acellular graft. In one preferred mode the infected cells can beintroduced into an autologous donor.

Another preferred embodiment of the present invention provides a methodfor obtaining transduced umbilical cord blood cells suitable for acellular enactment procedure. The method includes infectinghematopoietic cells from umbilical cord blood with areplication-defective recombinant retrovirus vector in the presence ofan effective immobilized amount of fibronectin and/or fibronectinfragments to increase the frequency of transduction of the hematopoieticcells by the retrovirus vector. The invention also includes viabletransduced cellular populations from umbilical cord blood obtainable bysuch a method, and cellular grafting methods which include introducingthe transduced cellular populations into an animal as a cellular graft.

In accordance with more specific aspects of the above-mentionedembodiments of the invention, the fibronectin or fibronectin fragmentutilized will contain a first amino acid sequence which provides theretroviral-binding activity of the Heparin-II-binding domain offibronectin, and a second amino acid sequence which provides thecell-binding activity of the CS-1 domain of fibronectin. The use ofthese two binding domains of fibronectin together has proven to verysignificantly enhance the transduction efficiency of the target cells bythe retrovirus.

Another preferred embodiment of the invention provides a method forproducing a construct for enhancing the frequency of transduction of apredetermined target cell by a retrovirus vector. The method includesthe step of covalently coupling a ligand which binds to said target cellto a polypeptide containing an amino acid sequence which provides theretroviral-binding activity of the Heparin-II binding domain offibronectin. The present invention also includes methods involving theutilization of these constructs to increase the frequency oftransduction of the predetermined target cells by a retrovirus vector,and to enactment procedures utilizing the transduced cells.

Another preferred embodiment of the invention provides a method forlocalizing an amount of a virus, comprising incubating a mediumcontaining the virus in the presence of an effective immobilized amountof fibronectin or fragments of fibronectin containing the viral-bindingactivity of the Heparin-II binding domain of fibronectin to localize anamount of the virus.

Still other preferred embodiments of the invention generally providetransduced viable hematopoietic and other cellular cultures containingsubstantially pure and/or immobilized fibronectin or fragments thereof,as well as kits for conducting retroviral-mediated DNA transfer intocells, as further described in the passages which follow.

A still further preferred embodiment of the invention provides a methodfor obtaining a transduced population of viable cells by a retrovirus,comprising infecting the cells with a retrovirus in the presence of aneffective immobilized amount of material such as polypeptide, whichamount of immobilized material includes a ligand which binds to thecells and a ligand which which binds the retrovirus, so as toco-localize the retrovirus and the cells and increase the transductionefficiency of the cells. It has further surprisingly been discoveredthat such processes are more advantageously conducted in the absence orat least the substantial absence of hexadimethrine bromide (alsoidentified as 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide),which heretofore has been used in gene transfer protocols for the desireto increase transduction efficiency. However, surprisingly, the presenceof hexadimethrine bromide has been discovered to reduce, rather thanincrease, transduction efficiency in the co-localization mediated genetransfer processes of the invention. Thus, more preferred processes ofthe invention are conducted in the absence of hexadimethrine bromide orother agents which increase transduction efficiency in similar protocolsconducted in the absence of the material for co-localization, forexample in corresponding co-culture protocols. Resultant improvedcellular populations and cellular grafting methods also form a part ofthe present invention.

It is an object of this invention to provide methods for efficientretroviral infection of mammalian cells.

It is a further object of this invention to provide methods for genetransfer with retroviral vectors which avoid the need for cocultivation.

It is a further object of the invention to provide improved methods andcellular cultures for autologous and/or allogeneic cellular grafting.

These and other objects, advantages, and features of the invention willbe readily apparent to the skilled artisan from the followingdescription.

DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic representation of a fibronectin molecule,including chymotryptic fragments.

FIG. 2 shows the infection efficiency of committed human progenitorcells in the presence of fibronectin fragments using the TKNEO vector,as further described in Example 1, infra.

FIG. 3 compares the infection efficiency of various committed humanhematopoietic progenitor cells in the presence of fibronectin fragmentsthereof using the TKNEO vector, as further described in Example 1,infra.

FIG. 4 compares the presence of hADA in mice engrafted with bone marrowcells transduced by (i) the coculture method (lanes 2-4), (ii)supernatant infection in the presence of immobilized fibronectinfragments (lanes 5-7), and supernatant infection on BSA (lanes 8 10), asfurther described in Example 7, infra. Controls for hADA are shown inlanes 1 and 12 and for murine ADA in lane 11.

FIG. 5 demonstrates retroviral binding to fibronectin fragments, asfurther described in Example 8, infra.

FIG. 6 demonstrates that retroviral binding to fibronectin fragments isdose-dependent, as further described in Example 8, infra.

FIG. 7 provides a schematic diagram illustrating various recombinantfibronectin fragments used in Examples 9-11, infra.

FIG. 8 shows retrovirus binding to various fibronectin fragments,including to several recombinant fragments, as described in Example 9,infra.

FIG. 9 demonstrates that heparin blocks retrovirus binding tofibronectin fragments, as described in Example 9, infra.

FIG. 10 shows the efficiency of retrovirus infection of murinehematopoietic cells in the presence of various fibronectin fragments, asfurther reported in Example 10, infra.

FIGS. 11 and 12 compare the presence of hADA in mice engrafted with bonemarrow cells transduced by (i) the coculture method, (ii) supernatantinfection on various fibronectin fragments, and (iii) supernatantinfection on BSA, as described in Example 11, infra.

FIG. 13 shows the structure of the α-chain of fibronectin and itsrelation to the recombinant fragments used in the Examples. Thefibronectin type I, II and III repeats are indicated and the type IIIrepeats numbered from 1 to 14. The three binding sites for cells aremarked as CELL for cell binding domain (CBD), HEPARIN for heparinbinding domain (HBD), and CS1 for the VLA-4 binding site CS1 formed bythe first 25 amino acids of the alternatively spliced IIICS region.

FIG. 14 shows the efficiency of retrovirus infection of NIH/3T3 cells inthe presence of various fibronectin fragments, as further reported inExample 12, infra.

FIG. 15 shows the efficiency of retrovirus infection of non-adherentHL60 cells in the presence of various fibronectin fragments, as furtherreported in Example 12, infra.

FIG. 16 shows the influence of low and high molecular weight heparin onretrovirus binding to fibronectin.

FIG. 17 shows the efficiency of retrovirus infection of various types ofprogenitor cells within a CD34+ cellular population in the presence of arecombinant fibronectin fragment, as discussed in Example 13, infra.

FIG. 18 shows the efficiency of retrovirus infection of HPP-CFC cells ina c-KIT+ cellular population in the presence of a recombinantfibronectin fragment, as discussed in Example 14, infra.

FIG. 19 demonstrates that the efficiency of retroviral infection ofNIH/3T3 cells decreases with increasing concentrations of hexadimethrinebromide, as discussed in Example 15, infra.

FIG. 20 demonstrates that the efficiency of retroviral infection ofclonogenic bone marrow cells decreases with increasing concentrations ofhexadimethrine bromide, as discussed in Example 15, infra.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to certain embodiments thereof andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations, further modificationsand such applications of the principles of the invention as illustratedherein being contemplated as would normally occur to one skilled in theart to which the invention relates.

As indicated above, the present invention provides methods forincreasing the frequency of transduction of viable cells by viruses suchas retroviruses. The invention also provides methods for efficient genetransfer into viable cells using recombinant retroviral vectors, methodsfor obtaining transduced cells, and methods and materials for achievingautologous and other cellular grafts.

One feature of the present invention is the discovery that fibronectin(FN), and fragments of fibronectin containing the CS-1 cell-adhesiondomain of FN, significantly enhance retroviral-mediated gene transferinto cells such as hematopoietic cells, e.g. committed progenitors andprimitive hematopoietic stem cells or long-term culture-initiating cells(LTC-IC), carrying a fibronectin receptor and thereby exhibiting thecapacity to bind to fibronectin or fragments thereof. Advantageously,this increased efficiency makes cocultivation with virus-producing cellsunnecessary. Other features of the invention capitalize on the discoveryof a viral-binding domain of fibronectin located within the Heparin-IIbinding domain. This viral-binding domain can be used to localize virusparticles in many applications, including for example in a broad rangeof constructs for delivering the virus to a target cell.

Recombinant viral vectors in accordance with certain preferred aspectsof the present invention contain exogenous DNA and are non-pathogenic,i.e. replication-defective. These vectors efficiently transfer andprecisely and stably integrate exogenous DNA into cellular DNA of hostcells such as animal cells, particularly mammalian cells. For example,in the present invention a nucleotide sequence including a run of basesfrom the coding sequence of the gene of interest can be incorporatedinto a recombinant retroviral vector under the control of a suitablepromoter to drive the gene, typically an exogenous promoter. In thisregard, the exogenous DNA can contain DNA which has either beennaturally or artificially produced, and can be from parts derived fromheterologous sources, which parts may be naturally occurring orchemically synthesized molecules, and wherein those parts have beenjoined by ligation or other means known to the art.

The exogenous DNA incorporated in the virus can be any DNA of interestfor introduction into the cells. For example, the exogenous DNA can codefor a protein such as ADA which is associated with a known disorder, oran antisense RNA, ribozyme or false primer (See, e.g. WO 90/13641published Nov. 15, 1990), for an intracellular antibody (See, e.g. WO94/02610 published Feb. 3, 1994), for a growth factor, or the like.

As indicated, the introduced nucleotide sequence will be under controlof a promoter and thus will be generally downstream from the promoter.Stated alternatively, the promoter sequence will be generally upstream(i.e., at the 5′ end) of the coding sequence. In this vein, it is wellknown that there may or may not be other regulatory elements (e.g.,enhancer sequences) which cooperate with the promoter and atranscriptional start codon to achieve transcription of the exogenouscoding sequence. The phrase “under control of” contemplates the presenceof such other elements as are necessary to achieve transcription of theintroduced gene. Also, the recombinant DNA will preferably include atermination sequence downstream from the introduced coding sequence.

Retroviral vectors that include exogenous DNA providing a selectablemarker or other selectable advantage can be used. For example, thevectors can contain one or more exogenous genes that provide resistanceto various selection agents including antibiotics such as neomycin.Representative vectors which can be used in the invention include forexample the N₂/ZipTKNEO vector (TKNEO) (titer: 1×10⁵ G418^(r) cfu/ml onNIH 3T3 cells), the ZipPGK-hADA vector, and the ZipPGK-mADA vector allas previously reported by Moritz et al. (1993) J. Exp. Med. 178:529. Inthe TKNEO vector, neo phosphotransferase sequences are expressed in thesense orientation (relative to the 5′ long terminal repeat-LTR) via theherpes simplex thymidine kinase promoter. This vector contains aselectable marker gene which provides neomycin resistance to facilitatethe identification of transduced cells. In the ZipPGK-hADA vector, thehuman ADA (“hADA”) cDNA is expressed in the sense orientation relativeto the 5′ LTR via the human phosphoglycerate kinase (PGK) promoter. Itcontains only one expressible genetic sequence and lacks a dominantselectable marker. The ZipPGK-mADA (PGK-mADA) vector is identical to theZipPGK-hADA vector except the human ADA cDNA has been replaced withmurine ADA (“mADA”) DNA. These and other viral vectors and techniquesfor their production are well known and their implementation and use inthe present invention will be well within the skills of those practicedin the art given the disclosure herein.

Viral vectors used in the invention exhibit the capacity to bind to anamino acid sequence of the Heparin-II binding domain of fibronectin,including that of human fibronectin. As discussed in the passages whichfollow, although the present invention is not limited by any theory, itis believed that co-localization of the virus and the target cell viabinding of the virus and cell to respective functional domainsfacilitates an enhancement in the transduction of the cell by the virus.In this regard, the capacity of a virus to bind to the amino acidsequence of the Heparin-II binding domain and thus to serve effectivelyin the invention can be readily ascertained using routine proceduressuch as those described in Examples 8 and 9 below. Generally speaking,these assays determine the extent to which virus particles are bound toimmobilized polypeptides containing the Heparin-II binding domain, so asto resist washing from the immobilized polypeptide matrix. Briefly, forinstance, a virus-containing supernatant can be incubated in a wellcontaining immobilized polypeptide including the fibronectin Heparin-IIbinding domain. The well is then extensively washed with physiologicsaline buffer, after which target cells to the virus are incubated inthe well to determine the level of infectious activity remaining in thewell. The reduction in infectious activity, or titer, relative to theinitial viral supernatant is assessed and compared to that of a similarcontrol run (e.g. using a BSA-coated well). A significantly higher titerremaining in the Heparin-II domain containing well as compared to thecontrol well signifies that the subject virus is suitable for use inaspects of the invention. To facilitate this screening procedure, theviral vector may contain a selectable marker gene, as discussed above.

Fragments of fibronectin for use in the invention can be of natural orsynthetic origin, and can be prepared in substantial purity fromnaturally-occurring materials, for example as previously described byRuoslahti et al. (1981) J. Biol. Chem. 256: 7277; Patel and Lodish(1986) J. Cell. Biol. 102:449; and Bernardi et al. (1987) J. Cell. Biol.105:489. In this regard, reference herein to a substantially purefibronectin or fibronectin fragments is intended to mean that they areessentially free from other proteins with which fibronectin naturallyoccurs. Substantially pure fibronectin or fibronectin fragments for usein the invention can also be recombinantly produced, for instance asgenerally described in U.S. Pat. No. 5,198,423 issued Mar. 30, 1993 toTaguchi et al. and assigned to Takara Shuzo Co., Ltd., Kyoto, Japan. Inparticular, the recombinant fragments identified in the Examples belowas H-271, H-296, CH-271, CH-296 and C-CS1, and methods for obtainingthem, are described in detail in this '423 patent. The C274 fragmentutilized in the Examples below was obtained as described in U.S. Pat.No. 5,102,988. These fragments or fragments from which they can beroutinely derived are available by culturing E. coli deposited at theFermentation Research Institute of the Agency of Industrial Science andTechnology, Japan as FERM P-10721 (H-296), FERM BP-2799 (C-277 bound toH-271 via methionine), FERM BP-2800 (C-277 bound to H-296 viamethionine), and FERM BP-2264 (H-271), as also described in U.S. Pat.No. 5,198,423. In addition, useful information as to fibronectinfragments utilizable herein or as to starting materials for suchfragments may be found in Kimizuka et al., J. Biochem. 110, 284-291(1991), which reports further as to the above-noted recombinantfragments; in EMBO J., 4, 1755-1759 (1985), which reports the structureof the human fibronectin gene; and in Biochemistry, 25, 4936-4941(1986), which reports on the Heparin-II binding domain of humanfibronectin. Fibronectin fragments which contain both the CS-1 celladhesion domain and the Heparin-II binding domain, for example asincluded in about a 30 or 35 kd fragment (30/35 FN) and in variousrecombinant fragments as reported in the Examples below, have been foundto significantly enhance the efficiency of gene transfer intohematopoietic cells in work thus far, and are preferred for use in theinvention. It will thus be understood that, broadly speaking, thefibronectin-related polypeptide or polypeptides utilized in theinvention will provide an amino acid sequence providing the cell-bindingactivity of the CS-1 cell adhesion domain of fibronectin as well as anamino acid sequence of the Heparin-II binding domain of fibronectinwhich binds the virus. The skilled artisan will recognize that thenecessary cell- and virus-binding activities can be provided both by thenative amino acid sequences of these functional fibronectin domains andby amino acid sequences which differ from the native sequences yet aresufficiently similar to exhibit the cell-binding and viral-bindingactivities. These similar amino acid sequences will exhibit substantialsequence homology to their corresponding native sequences, and caninclude those in which amino acids have been deleted, substituted forand/or modified while nonetheless providing an amino acid sequence withthe desired cell-binding or viral-binding characteristic.

In this regard, the pertinent biotechnological arts have advanced to astate in which the deletion, substitution, addition or othermodification of amino acids in the subject functional domains can beroutinely performed. The resulting amino acid sequences can then beroutinely screened for the desired cell-binding or viral-bindingactivity. For example, viral-binding activity of mutant or modifiedforms of the Heparin-II-binding domain of fibronectin can be screened asgenerally discussed above and more specifically below in Examples 8 and9, using virus incubation, wash, and viral titer assays to determine theretention of infectiousness compared to a control. Given the teachingsprovided herein, these binding assays will represent but routineexperimentation to those working in this field.

Cell-binding to modified or mutant forms of the CS-1 cell adhesiondomain of fibronectin, or to other cell-binding polypeptides, canlikewise be assayed using conventional procedures. For example, suchprocedures include those described in Nature 352: 438-441 (1991).Briefly, the cell-binding polypeptide is coated on plastic dishes andthe cell population to be assayed is overlayed in medium for 30 minutesto 2 hours. After this incubation period, cells non-adherent to theprotein are retrieved, counted and assayed for viability. Cells adherentto the polypeptide are also retrieved using trypsin or cell dissociationbuffer (e.g. Gibco), counted and viability tested. In some cases, forexample for hematopoietic colony forming cells, the cells are furthercultured for an additional 12-14 days to ascertain the colony formingcharacteristics of the cells. The percentage of adherent cells is thencalculated and compared to standard to a standard control such as bovineserum albumin (BSA) coated plastic dishes. Substantial binding of thetarget cells to the assayed polypeptide provides an indication that thepolypeptide/cell combination is suitable for the invention, and thepolypeptide can be coupled to the retroviral binding fragment fromfibronectin to produce a construct of the invention for enhancing theinfection of the target cells by the viral vector.

Pursuant to more specific aspects of the invention, the viral-bindingpolypeptide utilized to enhance transduction by retroviral vectors willcomprise (i) a first amino acid sequence which corresponds to the Ala¹⁶⁹⁰-Thr.¹⁹⁶⁰ of the Heparin-II binding domain of human fibronectin,which is represented by the formula (Seq. I.D. #1): Ala Ile Pro Ala ProThr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp ThrPro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro Lys Glu LysThr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser Ser Val Val ValSer Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu Lys AspThr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn Val SerPro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile SerTrp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala Val Pro AlaAsn Gly Gln Thr Pro Ile Gln Arg Thr Ile Sys Pro Asp Val Arg Ser Tyr ThrIle Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu AsnAsp Asn Ala Arg Ser Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp AlaPro Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser TrpGln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Sys Pro GlySer Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr Glu Ala ThrIle Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Tyr Val Ile Ala Leu LysAsn Asn Gln Lys Ser Glu Pro Leu Ile Gly Arg Lys Lys Thr;

-   -   or a sufficiently similar amino acid sequence thereto to exhibit        the ability to bind the retrovirus;

and (ii) a second amino acid sequence which corresponds to one portionof the IIICS binding domain of human fibronectin (the CS-1 cell bindingdomain); which is represented by the formula (Seq. I.D. #2): Asp Glu LeuPro Gln Leu Val Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu AspVal Pro Ser Thr;

-   -   or a sufficiently similar amino acid sequence thereto to exhibit        the ability to bind hematopoietic cells such as primitive        progenitor and/or long term repopulating (stem) cells.

As mentioned previously, it will be understood that certainmodifications and/or mutations of these native sequences are possiblewithin the practice of the present invention, so long as the resultingamino acid sequence is sufficiently similar to the native sequence toexhibit the ability to bind the virus (in the case of theHeparin-II-binding domain) and the ability to bind the target cells (inthe case of the CS-1 domain).

One aspect of the invention provides a method of somatic gene therapywhich involves in vitro cellular therapy and subsequent transplantationof target cells into a host, also known as “enactment” of the host withthe transduced target cells. Hematopoietic or other cells, for instancestem cells isolated from bone marrow or peripheral blood, embyronic stemcells, or cells otherwise characterized as CD34+ and/or C-kit+, can becollected from a human or other mammalian animal source using standardprotocols. For example, the hematopoietic cells can be collected frombone marrow or peripheral blood of a human donor or from human fetalumbilical cord blood. Once collected, the hematopoietic cells canoptionally be treated by standard techniques to enrich them in stemcells and/or primitive progenitor cells. The hematopoietic cells canthen be suitably incubated, for instance on tissue culture plates.Optionally during this period, adherent-negative low density mononuclearcells can be prestimulated prior to retroviral infection. Prestimulationas known in the art and as used herein refers to the process of exposingcells to growth stimulating factors before exposure to retroviruses.Such prestimulation has proven to improve the transduction ofhematopoietic cells by retroviruses.

Subsequent to prestimulation, the cells can be harvested and incubatedwith fibronectin or fragments thereof as described herein which enhancethe frequency of cellular transduction by retroviruses. Preferably, thecells are incubated with purified and/or insoluble, e.g., immobilizedfibronectin or fibronectin fragments. The cells can then be infectedwith the recombinant virus, for instance a retrovirus containing a genefor correcting an enzyme or other protein deficiency or abnormality inthe cells, in the presence of an amount of the fibronectin orfibronectin fragment effective to increase the frequency of transductionof the cells by the virus. The resulting transduced hematopoietic cellscan then be conventionally introduced, e.g. intravenously, into ananimal cellular graft recipient, preferably an autologous donor but alsoincluding allogeneic transplants, the latter especially where umbilicalcord blood cells are used for the graft as discussed below.

Methods of the invention can be used in gene marking or gene therapyprotocols for a variety of disorders including bone marrow disorders,including for example cancers, leukemias, disorders involving proteindeficiencies or abnormalities, and therapies for modifying hematopoieticcells to improve resistance to other therapeutic protocols such aschemotherapy. Representative disorders with which the invention may beused thus include ADA deficiency, e.g. ADA-deficient SCID, pediatricacute myelogenous leukemia (AML), neuroblastoma, and adult AML and acutelymphocytic leukemia (ALL).

In one particularly preferred embodiment of the invention, the cellsutilized for a cellular graft are obtained from human umbilical cordblood. Thus, human umbilical cord blood can be collected and enriched inviable primitive hematopoietic progenitor and/or stem cells, for exampleby obtaining an adherent-negative, low density, mononuclear cellpopulation. This population is then optionally prestimulated, andincubated in the presence of a retroviral vector and immobilized and/orpurified fibronectin or fibronectin fragments, to enhance thetransduction efficiency of the cells by the vector. In this regard ithas been found that the transduction of the primitive hematopoieticand/or stem cells from umbilical cord blood is greatly enhanced in thepresence of the fibronectin or fibronectin fragments, even thoughfibronectin does not constitute part of the ECM in cord blood and eventhough primitive progenitor and stem cells from cord blood have beencharacterized as different from those from bone marrow. In particular,the cord blood stem cell has been characterized as CD34 ⁺, HLA-DR⁺,whereas the stem cell from bone marrow has been characterized as CD34 ⁺,HLA-DR.⁻. The discovery that primitive progenitor cells from umbilicalcord blood are effectively transduced in an enhanced fashion in thepresence of the fibronectin or fibronectin fragments enables the use ofa convenient and highly stem-cell-enriched source of hematopoieticcells. Moreover, evidence of successful enactment of numerous patientswith allogeneic transplants of cord blood enriched for primitiveprogenitor and stem cells, makes cord blood a highly preferred sourcefor hematopoietic cells. See, Kohli-Kummer et al., Brit. Heaematol.85:419-422 (1993); Broxmeyer et al., Blood Cell 17:313-329 (1991);Gluckman et al., Br. J. Heaematol. 45:557 (1980); Heidelberg:Springer-Verlag pp. 60-68 (1989); Wagner et al., Blood 79:1874-1881(1992); and Wagner et al., Blood 82-86a (Abstract).

If desired, harvested transduced hematopoietic or other cells can betested for transduction efficiency and gene expression. For instance,the significant improvements in retrovirus-mediated gene transferprovided by the invention are demonstrated in the specific Examplesbelow, which describe several tests demonstrating high infection andgene transfer efficiency by retroviruses in the presence of fibronectinor effective fibronectin fragments. In particular, murine hematopoieticcells infected with PGK-hADA retrovirus expressed high levels of thetransferred ADA cDNA. Similarly, individual PGK-mADA virus infectedhuman progenitor colonies expressed murine ADA levels up to 10-foldhigher than the endogenous human ADA protein. Therefore, to stringentlyanalyze transfer efficiency, progenitor colonies were consideredtransduced only if expression of the transferred mADA was equal to orgreater than endogenous human ADA levels. High levels of expression ofneo from the TKNEO vector were detected by G418 drug resistance, as anassay for neophosphotransferase (the neo gene product) activity.

As indicated above, methods of the present invention are advantageouslyconducted without the need for cocultivation in the presence ofretroviral producer cells. Thus, in accordance with one aspect of theinvention, the retroviral-mediated gene transfer can be carried out inthe substantial absence of cells other than the target hematopoietic orother cells. For example, producer cells containing the retroviralvector plasmid can be cultured and supernatant collected. Theretroviral-containing supernatant can then be utilized to infect thehematopoietic cells in the presence of the fibronectin and/orfibronectin fragments, which are preferably in immobilized form, e.g.coated on a substrate upon which the infection is carried out orotherwise in contact with the medium for infection. In this regard, anyproducer cells which produce high-titer helper-free retroviruses arecontemplated as suitable for use in the invention. These include, forexample, packaging cells such as Psi-2, C2, PA12, PA317, and GP+envAM12,as well as many other packaging cell lines known in the art.

In accordance with other features of the invention, the strong virusbinding to amino acids within the Heparin-II binding domain offibronectin may be used for constructing delivery systems for viraltherapy across a broad range of cell types. To this end, a polypeptideincluding the retrovirus binding domain from fibronectin may becovalently coupled to any ligand which gives this construct specificityfor the target cells. This approach will circumvent the prior necessityof constructing specific retrovirus cell lines for each target cell(Kasahara, N., A. M. Dozy, and Y. W. Kan., Science Vol. 266, pp.1373-1376 (1994) and Valsesia-Wittmann, S., A. Drynda, G. Deleage, M.Aumailley, J. M. Heard, O. Danos, G. Verdier, and F. L. Cosset, J.Virol., Vol. 68, pp 4609-4619 (1994)). The specificity of the targetingconstruct may be provided by employing ligands including for example 1)cell adhesive protein, 2) hormones or cytokines, 3) monoclonalantibodies to the target cells, 4) carbohydrates which bind the targetcells (G. Ashwell, et al., Annu. Rev. Biochem., Vol. 51, pp. 531-554(1982)), 5) metabolites for the target cells, or 6) functionalpolypeptides which bind the target cells. The efficiency of theconstruct for gene delivery may be improved by including severalHeparin-II virus binding domains and therefore increasing the amount ofviral particles delivered to the target cells. For example, thecell-binding domain of human fibronectin which corresponds toPro¹²³⁹-Ser^(1515,) as described in U.S. Pat. No. 5,198,423, has beenshown to bind to cells including BHK and B16-F10 cells (Kimizuka et al.,J. Biochem. Vol. 110, pp. 285-291 (1991)). In addition, the Heparin-IIdomain itself has been shown to bind to fibroblasts, endothelial cells,and tumor cells. These polypeptide sequences may be coupled to theretrovirus binding domain from fibronectin to target predetermined cellsfor infection by retrovirus.

Exemplary applications in the hematopoietic system also include aconstruct of erythropoietin or G-CSF coupled to the retrovirus bindingdomain of fibronectin for targeting highly specific erythroid orgranulocytic precursor cells, respectively. Another common applicationin accordance with the present invention will be to combine theretrovirus binding domain or domains with a ligand which specifically orpredominantly binds to malignant cells. For example, it has been shownthat in vitro and even in vivo growth of breast carcinoma cells can beinfluenced employing substances binding to receptors on the target cellslike luteinizing hormone releasing derivatives, Emons, G. et al., Hum.Reprod. 9:1364-1379 (1994), oestrogens, Tolcher, A. W., Oncol. 8:39-43(1994), or anti-oestrogens, Howell, A. et al., Lancet 345:29-30 (1995),progestogens or anti-progestogens, Klijn. F. G. et al, Hum. Reprod. 9Suppl. 1:181-189 (1994); Griffiths, K. et al, Semin. Oncol. 21:672-687(1994), which will serve as ligands in constructs of the inventioncontaining one or more virus binding domains from fibronectin. Asfurther examples, thyroid (cancer) cells may be targeted highlyspecifically by using constructs with Jodid, and liver (cancer) cellsmay by targeted by constructs containing HDL or parts thereof. Finally,constructs of monoclonal antibodies and the retrovirus-binding domain offibronectin will allow the targeting of any cell and organ against whichan antibody is available. A broad range of mammalian cell types are thustargetable by capitalizing upon the ability of the retrovirus bindingdomain of fibrobnectin to bind and localize viral vectors.

Accordingly, another preferred embodiment of the invention involves thepreparation of construct which can be used to enhance the viraltransduction of a target cell. The viral-binding amino acid sequence ofthe Heparin-II-binding domain of fibronectin is coupled to a ligand towhich the target cell binds. As discussed above, the ligand may be, forexample, a polypeptide from fibronectin or from another protein(including a cell adhesive protein, for example laminin, collagen,vitronectin, osteopontin or thrombospondin), a hormone, a metabolite, anantibody (including monoclonal antibodies), or any other ligandexhibiting the capacity to bind; preferably with specificity, to thetarget cell. The resulting overall construct can be used in immobilizedform in a fashion similar to that used for the fibronectin polypeptidesspecifically exemplified in the Examples below.

Such constructs and cell-targeting approaches may be utilized in vitroas discussed above, and also in in vivo targeting of retroviruses,taking into account various factors such as the stability andspecificity of the construct and the retrovirus construct interactionunder physiological conditions. The specificity may also be improved bymodifying the delivery system to localize delivery of the construct tothe target cells, for instance catheterizing the portal vein fortargeting liver cells.

Another aspect of the invention relates to the discovery that thetransduction processes of the invention, which involve co-localizationof the virus and the cells; are more advantageous when conducted in theabsence or substantial absence of hexadimethrine bromide. Hexadimethrinebromide (commercially available under the name Polybrene® is apolycationic substance which has been extensively used inretroviral-mediated gene transfer protocols for the purpose of improvingthe tranduction efficiency by the retrovirus. Nonetheless, it has beendiscovered that the presence of hexadimethrine bromide inco-localization enhanced gene transfer protocols such as those describedherein significantly reduces transduction efficiency. Thus, improvedprocesses of the invention are conducted in a medium at leastsubstantially free from hexadimethrine bromide (i.e. containing no morethan about 1 μg/ml hexadimethrine bromide) and more preferably in theabsence of hexadimethrine bromide. Such processes provide preferredcellular compositions of the invention, which include substantiallyretroviral-transduced viable cellular populations which aresubstantially free from both retroviral producer cells andhexadimethrine bromide. In this regard, substantially transduced viablecellular populations as used herein is intended to mean that at leastabout 20% of the cells in the population have been tranduced by aretrovirus. More preferred populations will have at least about 50%transduced cells, and most preferably at least about 75% transducedcells. Preferred cellular compositions in accordance with this aspect ofthe invention will include hematopoietic cells, and more preferably willinclude hematopoietic cellular populations which are enriched inprimative progenitor and stem cells. Generally speaking, advantageousprocesses of the invention can thus be conducted without the presence ofpolycationic or other agents which, in corresponding retroviralinfection protocols (e.g. co-culture) without the fibronectin fragmentor other material for co-localization, lead to an increase intransduction efficiency, but which agents reduce the transductionefficiency in the presence of the material for co-localization.

It is contemplated that highly convenient retroviral-mediated DNAtransfer will be carried out utilizing kits specially designed topractice methods of the invention. Accordingly, another aspect of theinvention provides kits which include an amount of the substantiallypure polypeptide or construct discussed above which enhances thetransduction of target cells by retroviruses, along with an artificialsubstrate upon which the retroviral infection can be carried out. Thepolypeptide or other construct can be provided separately or coated uponthe artificial substrate. In the case of infection protocols for humanhematopoietic cells the kits will also include hematopoietic cell growthfactors for cell prestimulation. In addition, the kits can include therecombinant retrovirus vectors as discussed above for the transduction.Generally speaking, the kits will include sterile packaging whichsecures these various kit components in spaced relation from one anothersufficient to prevent breakage of the components during handling of thekit. For example, it is a common practice to utilize molded plasticarticles having multiple compartments or areas for holding the kitcomponents in spaced relation.

In order to promote a further understanding and appreciation of theinvention, the following specific Examples are provided. It will beunderstood that these examples are illustrative and not limiting innature.

EXAMPLE 1 Gene Transfer into Bone Marrow Cells Using TKNEO

1.1. Preparation of Virus-Supernatant

GP+EnvAM 12 producer cells (see Markowitz et al. (1988) Virology167:400) containing retroviral plasmid TKNEO vector were cultured inIscove's Modified Dulbeccos Medium (IMDM, Gibco, Gaithersburg, Md.)containing 10% fetal calf serum (FCS, Hyclone, Logan, Utah) and 100units/ml penicillin and 100 microgram/ml streptomycin (P/S, both Gibco).Virus containing supernatant was collected by adding 10 ml of IMDMcontaining 20% FCS to confluent plates overnight. Harvested medium wasfiltered through 0.45 micron filters (Gelman Sciences, Ann Arbor, Mich.)and stored at −80° C. until used.

1.2. Preparation of Fibronectin Fragments

FN was purified from human plasma (Lifesource, Glenview, Ill.) aspreviously described in Ruoslahti et al., Methods Enzymol. 82:803-831(1982), except that the gelatin-agarose column was washed with 1M ureaprior to elution of FN with 4M urea. Purified FN was dialyzedextensively at 4° C. against 10 mM3-(cyclohexylamino)-1-propane-sulfonic acid (CAPS), 150 mM NaCl, 2 mMCaCl.sub.2 pH 11.0 and stored in aliquots at −80° C. The chymotrypticcell binding domain (CBD) (CS-1) and Heparin-II binding fragments of FNwere purified as previously described (Ruoslahti et al. (1982), supra,Patel and Lodish, J. Cell. Biol. 102, pp. 449-456 (1986), and Bernardiet al., J. Cell. Biol. 105, pp. 489-498 (1987). Three majorheparin-binding fragments (30 kD, 35 kD, and 42 kD) were obtained in the1M NaCl eluate from the heparin-agarose column. To further purify theseheparin-binding fragments, the 1M NaCl eluate was dialyzed overnight at4° C. against lOmM Tris-HCl, pH 7.0 and passed over an anion exchangecolumn (2 ml DEAE sepharose fast flow (Pharmacia Fine Chemicals,Uppsala, Sweden)/mg of protein) that had been equilibrated with 10 mMTris-HCl pH 7.0. The 30/35 kD fragments were collected in the unboundfraction while the 42 kD fragment was eluted from the column with 100 mMNaCl. From 500 mg of FN, approximately 26 mg of the 30/35 kD fragmentsand 4 mg of the 42 kD fragment were obtained. The 42 kD fragment, butnot the 30/35 kD fragments, were recognized by an antibody against thefibrin-binding domain, as determined by western blotting technique.Also, the 42 kD fragment binds to a fibrin-sepharose affinity column.

For use in the infection protocol, fibronectin fragments wereimmobilized on 35 or 100 mm petri dishes (Falcon, Lincoln Park, N.J.) ata concentration of 75 pmol/cm² as described by Patel and Lodish (1986),supra. Control plates were coated in analogous fashion with 2% (FN-free)bovine serum albumin (BSA, Boehringer Mannheim, Mannheim, Germany).

1.3. Retroviral Infection Protocol

Bone marrow samples from healthy adult donors were collected in tubescontaining sterile, preservative-free sodium sulfate heparin accordingto protocols approved by the Institutional Review Board of IndianaUniversity School of Medicine. Low density mononuclear cells wereprepared by centrifugation on Ficoll-Hypaque (density 1.077 g/ml,Pharmacia, Piscataway, N.J.) for 45 minutes at 25° C. Plastic adherentcells were removed from low density bone marrow cells by an additionalincubation on tissue culture plates for 4-16 hours at 37° C. in 5% CO₂in IMDM with 2-% FCS.

Adherent-negative low density mononuclear cells were prestimulated priorto retroviral infection as described previously by Luskey et al. (1992)Blood 80:396, for 48 hours at 37° C. and 5% CO₂ in IMDM containing 20%FCS, 100 U/ml rhIL-6, 100 ng/ml rhSCF (both Amgen, Thousand Oaks,Calif.), and P/S at a cell density of 1×10⁶ cells/ml in petri dishes.Prestimulated cells were harvested by vigorously pipetting to removecells loosely adherent to the plastic.

Prestimulated cells (5×10⁵ cells/ml) were incubated for 6 hours onplates coated with BSA (control plates) or fibronectin or fragmentsthereof (subjected to UV radiation to better adhere the proteins to theplastic plate) and then infected with virus supernatant in the presenceof growth factors (as above) and 7.5 micrograms/ml polybrene (AldrichChemical, Milwaukee, Wis.). Virus supernatant was replaced (includinggrowth factors and 5.0 microgram/ml polybrene) after 2 hours and cellswere incubated for an additional 12 to 24 hours. Non-adherent cells werere-added with each media change.

Following the infection protocol, non-adherent cells were decanted andadherent hematopoietic cells were collected from the cultures using CellDissociation Buffer (CDB) (enzyme free/PBS based, Gibco) according tothe manufacturer's instructions. The adherent cells were added to thenon-adherent fraction, washed twice and counted. Harvested cells wereeither plated in clonogenic methylcellulose progenitor assays or longterm bone marrow cultures.

1.4. Long Term Bone Marrow Cultures

LTC-IC (human stem cell) assays were performed according to previouslydescribed methods with slight modifications. Sutherland et al. Blood74:1563 (1989). Briefly, 0.5-1×10⁶ infected cells were seeded in longterm bone marrow cultures (LTMC) on confluent, pre-irradiated (as above)allogenic human bone marrow fibroblasts (BMF) in 5 ml IMDM containing10% FCS, 10% horse serum (Sigma) and P/S, 1×10⁻⁵ M hydrocortisone(Upjohn, Kalamazoo, Mich.), and 320 mosmol sodium chloride in 6 welltissue culture plates (Costar, Cambridge, Mass.). LTMC were incubated at33° C. in 5% CO₂ and fed weekly by removal of 50% of the media andnon-adherent cells. After five weeks, LTC-IC cultures were sacrificed byusing CDB to remove adherent hematopoietic cells from BMF. Bothnon-adherent and adherent hematopoietic cells were combined and platedin methylcellulose to obtain colonies derived from LTC-IC.

1.5. Clonogenic Methylcellulose Assays

Methylcellulose assays were performed as previously described by Toksozet al. Proc. Nat. Acad. Sci., USA, Vol. 89, p 7350 (1992), with minormodifications. Briefly, 2-5×10⁴ infected adult bone marrow cells wereplated with 5 units/ml erythropoietin (Epo, Amgen), 100 ng/ml rhSCF, 10ng/ml rhIL-3 (Genzyme, Cambridge, Mass.) in 1 ml of 2.4% IMDMmethylcellulose (Fluka, Ronkonkoma, N.Y.) containing 25% FCS, 10% humanplasma, 10⁻⁵ M beta-mercaptoethanol (Sigma), and P/S. Cultures wereincubated at 37° C. in 5% CO₂/95% air and colonies (>50 cells) werescored by viewing on an inverted microscope on day 13 as CFU-GM(containing granulocytes and macrophages), CFU-Mix (containing myeloidand erythroid elements), or BFU-E (containing only erythroid elements).

1.6. Analysis of Retroviral Infection

Efficiency of infection with the TKNEO virus was analyzed by determiningthe percent of methylcellulose colonies resistant to 1.5 mg/ml (drypowder, Gibco) G418 on day 13. Mock infections were performed in eachexperiment by incubating bone marrow on the GP+EnvAM 12 packaging linemaking no recombinant virus. Culture of these mock infected cells with1.5 mg/ml G418 consistently demonstrated <1% background colonies.

1.7. Gene Transfer Efficiency into Committed Progenitor Cells

Transduction efficiency was compared by infecting bone marrow cellswhile plated on 30/35 FN- or BSA-coated dishes. No difference in thenumber of colonies obtained after infection without selection wasobserved between these conditions. FIG. 2 demonstrates the percentage ofG418. supr colonies after infection. A higher percentage of G418rcolonies was noted on 30/35 FN from all types of progenitors, includingthose derived from lineage-restricted (BFU-E and CFU-GM) as well asmultilineage (CFU-Mix) progenitor cells. Infection into all committedprogenitors was increased 9-fold on 30/35 FN versus BSA.

1.8. Gene Transfer Efficiency into Long Term Culture-Initiating Cells

Gene transfer into LTC-IC was assessed using the TKNEO vector. Genetransfer into LTC-IC derived colonies was only detected after infectionon 30/35 FN (16% G418′ vs 0% G418^(r) colonies, 30/35 FN vs BSA).

b 1.9. Specificity of 30/35 FN Effects on Infection Efficiency ofHematopoietic Cells

To determine the specificity of increased gene transfer efficiency seenon 30/35 FN, infection with TKNEO was performed on plates coated withBSA, 30/35 FN, intact fibronectin, a 115 kd FN fragment containing thecentral cell-binding domain (CBD) containing the RGDS tetrapeptidesequence, and a 42 kd C-terminal FN fragment (42FN) characterized by theHeparin-II binding domain but lacking the CS-1 sequence (FIG. 1).Infection on BSA yielded 3±1% G418^(r) BFU-E, 1±1% G418^(r) CFU-GM, and0±0% G418^(r) CFU-MIX. No significant increase in the proportion ofG418^(r) colonies were noted on CBD, while slightly higher infection ofBFU-E (6.0±1%) were seen on 42 FN (FIG. 3). However, intact FN promotedincreased gene transfer into all committed progenitors. The percentageof G418.sup.r colonies after infection on intact FN were less than on30/35 FN in all lineages, including BFU-E (16±2 vs. 24±4%), CFU-GM (5±2vs 20±4%) and CFU-Mix (6±1 vs 9±1; intact FN vs 30/35 FN, respectively.

EXAMPLE 2 Gene Transfer into Bone Marrow Cells Using PGK-mADA

2.1. General Procedures

PGK-mADA virus supernatant was prepared as described for TKNEO inExample 1. Chymotryptic fragments of fibronectin (FIG. 1) were preparedas previously described in Example 1 and the retroviral infectionprotocol of Example 1 was followed. LTC-IC (human stem cells) assays andMethylcellulose assays were performed according to Example 1.

2.2 Analysis of retroviral infection

Efficiency of infection with the PGK-mADA vector was determined byprotein analysis using ADA isoenzyme electrophoresis. Analysis ofindividual progenitor colonies was performed as previously described byMoritz (1993) and Lim et al. (1989) Proc. Natl. Acad. Sci., USA, Vol.86, p 8892. To stringently analyze transfer efficiency, only coloniesexpressing mADA at the same or a higher level than endogenous human ADAwere considered transduced. For analysis of pooled colonies, coloniespicked out of methylcellulose culture were combined in 1.5 ml microtubes(Rainin, Woburn, Mass.), washed with warm medium and phosphate bufferedsaline (PBS), centrifuged and stored at −20° C. For ADA analysis, cellswere lysed in 5 microliter of lysis buffer by repeated freezing-thawingcycles and isoenzyme electrophoresis was performed as previouslydescribed.

2.3. Gene Transfer Efficiency into Committed Progenitor Cells

Transduction efficiency was compared by infecting bone marrow cellswhile plated on 30/35 FN- of BSA-coated dishes. No difference in thenumber of colonies obtained after infection without selection wasobserved between these conditions. As shown in Table 1, infectionefficiency into all committed progenitors was substantially increased on30/35 FN vs BSA. As expected with the high titer (˜1×10⁷ virons/ml)vector, the transduction efficiency of committed progenitors wasextremely high. Referring to Table I, infection of bone marrow on 30/35FN with PGK-mADA yielded nearly 100% transduction of committedprogenitors in two separate experiments. TABLE 1 Infection efficiency ofcommitted human progenitor cells on fibronectin 30/35 fragments usingthe PGK-mADA vector EXPERIMENT BSA 30/35 FN Exp 1 1/18* 13/14 Exp 2 2/1312/13*number of MADA expressing colonies/total colonies analyzed2.4. Gene Transfer Efficiency into Long Term Culture-Initiating Cells

In four independent experiments performed with PGK-mADA a significantproportion of progenitor colonies derived from 5 week old LTMC (i.e.LTC-IC derived colonies) expressed the transferred murine ADA gene.Expression ranged from 2/12 (17%) to 6/6 (100%) of analyzed colonies(Table 2). Expression of the introduced mADA gene exceeded or at leastequaled the amount of endogenous human ADA activity in all coloniesconsidered positive. Infection efficiency for PGK-MADA was higher thanfor TKNEO. As shown in Table 2, infection of bone marrow on 30/35 FNwith PGK-MADA yielded nearly 100% transduction of committed progenitorsin two separate experiments. TABLE 2 Infection efficiency of human longterm culture initiating cell (LTC-IC) using the PGK-mADA vectorEXPERIMENT BSA 30/35FN Exp 1 0/14* 10/19 Exp 2 N/A  2/12 Exp 3 0/4  3/5Exp 4 0/4  6/6 Total 0/22 21/42*number of mADA positive colonies/total colonies analyzed;N/A; not analyzed2.5. Specificity of 30/35 FN Effects on Infection Efficiency ofHematopoietic Cells

Gene transfer efficiency into LTC-IC was increased on 30/35 FN. Due tothe relatively small size of these secondary LTC-IC derived colonies,the ability to perform protein analysis on single colonies was limited.After infection with PGK-mADA on BSA, intact fibronectin and 42 FN 0/6,0/4, and 0/3 LTC-IC-derived colonies, respectively, demonstratedexpression of murine ADA, while 3/5 LTC-IC-derived colonies infected on30/35 FN expressed mADA. In addition, when multiple LTC-IC-derivedcolonies were pooled before analysis in two additional experiments, mADAexpression was detected only after infection on 30/35 FN and to a lesserdegree on intact FN, but not on 42FN or BSA.

EXAMPLE 3 Gene Transfer into Bone Marrow Cells Using PGK-hADA

3.1. General Procedure

PGK-hADA virus supernatant is prepared as described for TKNEO inExample 1. Chymotryptic fragments of fibronectin (FIG. 1) are preparedas previously described in Example 1 and the retroviral infectionprotocol of Example 1 was followed. LTC-IC and methylcellulose assayswere performed as described in Example 1.

3.2. Analysis of Retroviral Infection

For analysis of pooled colonies, colonies picked out of methylcelluloseculture are combined in 1.5 ml microtubes (Rainin, Woburn, Mass.),washed with warm medium and PBS, centrifuged and stored at −20° C. ForADA analysis, cells are lysed in 5 microliter of lysis buffer byrepeated freezing-thawing cycles and isoenzyme electrophoresis isperformed as previously described.

EXAMPLE 4 Gene Transfer into Cord Blood Cells Using TKNEO

4.1. General Procedure

TKNEO virus supernatant and chymotryptic fragments of fibronectin(FIG. 1) were prepared as previously described in Example 1. Theretroviral infection protocol in Example 1 was followed except thatumbilical cord blood from normal, full term newborn infants wascollected in tubes containing heparin according to protocols approved bythe Institutional Review Board of Indiana University School of Medicine,and used instead of the bone marrow cells. LTC-IC (human stem cell) andmethylcellulose assays were performed according to Example 1.

b 4.2. Gene Transfer Efficiency into Committed Progenitors

Infection on FN30/35 was more than four times increased compared to BSAin three separate experiments (Table 3). TABLE 3 Infection Efficiency ofCord Blood Progenitor Cells Using 30/35 FN Fragment and TKNEO Vector BSA12 ± 17 30/35 55 ± 16

EXAMPLE 5 Gene Transfer into Cord Blood Cells Using PGK-mADA

5.1. General Procedure

PGK-mADA virus supernatant and chymotryptic fragments of fibronectin(FIG. 1) were prepared as previously described in Example 1. Theretroviral infection protocol in Example 1 was followed except that cordblood from normal, full term newborn infants was collected in tubescontaining heparin according to protocols approved by the InstitutionalReview Board of Indiana University School of Medicine. LTC-IC andmethylcellulose assays were performed according to Example 1.

5.2. Gene Transfer Efficiency into Long-Term Culture Initiating Cells

Using the higher titer PGK-mADA vector, analysis of LTC-IC-derivedcolonies demonstrated high level expression of the introduced mADA cDNAonly from cultures established from cord blood infected usingsupernatant on FN30/35. Little expression of mADA was detected inLTC-IC-derived colonies infected in BSA control plates.

The results shown in Examples 4 and 5 demonstrate that improvedinfection efficiency using FN30/35 can also be achieved when using cordblood progenitor and stem cells.

EXAMPLE 6 Gene Transfer into Cord Blood Cells Using PGK-hADA

PGK-hADA virus supernatant and chymotryptic fragments of fibronectin(FIG. 1) are prepared as described for TKNEO in Example 1. Theretroviral infection protocol in Example 1 is followed except that cordblood from normal, full term newborn infants is collected in tubescontaining heparin according to protocols approved by the InstitutionalReview Board of Indiana University School of Medicine, and used insteadof the bone marrow cells. LTC-IC and methylcellulose assays areperformed according to Example 1.

EXAMPLES 7-11

Retroviral vectors and producer cell lines For Examples 7-11.

For Examples 7-11, two retrovirus-producing packaging cell lines wereemployed: the ecotropic GP+E86 (Markowitz, D., S. Goff, and A. Bank, J.Virol., Vol. 62, pp 1120-1124 (1988)) and the amphotropic GP+envAM12cell lines (Markowitz, D., S. Goff, and A. Bank, Virology, Vol. 167, pp400-406 (1988)), respectively. The retroviral vectors and producerclones used in studies described here are listed in Table 1. TABLE 4VECTOR PRODUCER/clone cDNA expressed PGK-hADA GP + E86/EPHA-5 humana ADAPM5neo GP + E86/EAL2a neo phosphotransferase, LAC-Z TKNeo GP + E86/TKNeoneo phosphotransferase PGK-mADA GP + EnvAM12/55/6 murine ADA

All cell lines were cultured in Dulbecco's modified Eagles medium (DME,Gibco, Grand Island, N.Y.) containing 10% fetal calf serum (FCS,Hyclone, Logan, Utah) and 100 units/ml penicillin and 100 .mu.g/mlstreptomycin (P/S, both Gibco) except for EAL2a cells which were grownin DME-F12 (Gibco) with 10% FCS plus P/S. Virus containing supernatantwas collected by adding 10 ml of alpha-minimal essential medium (αMEM,Gibco) for murine cells or Iscove's Dulbecco Medium (IMDM, Gibco) forhuman cells each containing 10% FCS plus P/S to confluent 10 cm platesovernight. Harvested medium was filtered through 0.45 micron filters(Gelman Sciences, Ann Arbor, Mich.) and stored at −80° until used.

EXAMPLE 7 Transduction of Primary Murine Hematopoietic Cells

7.1. Experimental

For studies with murine cells, bone marrow was harvested from femurs andtibiae of 6 to 8 week old C3H/HeJ mice 2 days following administrationof 150 mg/kg 5-fluorouracil (SoloPack Laboratories, Franklin Park, Ill.)(Lim, B., J. F. Apperley, S. H. Orkin, and D. A. Williams, Proc. Natl.Acad. Sci. USA, Vol. 86, pp 8892-8896 (1989)). Cells were prestimulatedat a concentration of 5×10⁵ cells/ml in IMDM/20% FCS plus P/S with 100ng/ml rat recombinant stem cell factor (rrSCF; Amgen, Thousands Oaks,Calif.) and 100 units/ml recombinant human interlukin-6 (rhIL-6; PeproTech Inc., Rock Hill, N.J.) for 48 hours (Luskey, B. D., M. Rosenblatt,K. Zsebo, and D. A. Williams, Blood, Vol. 80, pp 396-402 (1992)).Subsequently, gene transfer efficiency with the PGK-hADA vector producedby EPHA-5 producer cells was compared using three different infectionprotocols: 1) supernatant infection; 2) supernatant infection on FN30/35; 3) cocultivation on EPHA-5 producer cells. Therefore, 100 mmbacterial dishes were coated with 2.5 μg/cm² FN 30/35 (equivalent to 75pmol/cm²) dissolved in 5 ml phosphate buffered saline (PBS; Gibco) for 1hour at room temperature under UV light with the dish lid open and foranother hour with the dish lid closed. After blocking with 2% bovineserum albumin (BSA, Fraction V; Boehringer Mannheim, Indianapolis, Ind.)for 30 minutes at room temperature, dishes were washed once with Hank'sBalanced Salt Solution (HBSS) supplemented with 2.5% (v/v) 1M Hepes(both Gibco). For supernatant infection, dishes were coated with BSAonly. 5×10⁶ prestimulated donor cells were incubated with 10 ml of virussupernatant obtained from EPHA-5 cells as described above supplementedwith 100 U/ml rhIL-6, 100 ng/ml hrSCF and 7.5 μg/ml polybrene.Non-adherent cells were collected and re-added with the fresh virussupernatant. For co-culture, EPHA-5 cells in 4 ml medium were incubatedwith 10 μg/ml mitomycin C for 2 hours at 37. degree. C., washed,trypsinized and seeded on 100 mm tissue culture dishes at aconcentration of 3×10⁶ cells in 10 ml αMEM/20% FCS plus P/S. The nextday, 5×10⁶ prestimulated bone marrow cells with 100 U/ml rhIL-6, 100ng/ml rrSCF and 4 μg/ml polybrene were added for 48 hours. Following theinfection protocol, non-adherent cells were decanted and adherenthematopoietic cells were collected from the cultures using CellDissociation Buffer (CDB) (enzyme free/PBS based, Gibco) according tothe manufacturer's instructions. The adherent cells were added to thenon-adherent fraction, washed twice, and suspended in approximately 1 mlof HBSS/Hepes. The total cells obtained from 5×10⁶ prestimulated cellswere injected into the tail veins of three recipient mice which had beensubjected to lethal total body irradiation (with 700 plus 400 cGy,¹³⁷Cs-source) (Luskey, B. D., M. Rosenblatt, K. Zsebo, and D. A.Williams, Blood, Vol. 80, pp 396-402 (1992)). The transduction ofhematopoietic stem cells was analyzed by examination of reconstitutedmice for the expression of the introduced human ADA cDNA. This ADAisoenzyme analysis was performed in transplanted mice by examiningperipheral blood cells for the presence of the hADA protein by celluloseacetate in situ enzyme analysis (Lim, B., D. A. Williams, and S. H.Orkin, Mol. Cell. Biol., Vol. 7, pp 3459-3465 (1987)). Examination wasperformed beginning 4 months post-transplant and was repeated monthly.

7.2 Results

Long-term bone marrow reconstitution of mice with geneticallymanipulated hematopoietic stem cells is generally accepted as adequateto determine the efficiency of stem cell transduction after a period of4 months post-transplant. Analysis of recipients of transduced bonemarrow after 7 months by isoenzyme analysis revealed that: 1) human ADAcDNA expression was present using either co-culture or supernatantinfection on FN 30/35 but absent in the group transplanted aftersupernatant infection without FN 30/35 and that; 2) the levels ofexpression were comparable for the co-culture and FN 30/35 groups. Asshown in FIG. 4, lanes 2 4, three mice transplanted with bone marrowtransduced by co-culture on EPHA-5 cells demonstrated easily detectablehuman ADA. Similar levels of human ADA were detected in three micetransplanted with hematopoietic cells transduced by supernatantinfection of FN 20/35 (FIG. 4, lanes 5-7). In contrast, no human ADA wasdetected in three mice transplanted with hematopoietic cells transducedby supernatant infection on BSA (FIG. 4, lanes 8-10). Controls for thelocation of human ADA are shown in lanes 1 and 12 and murine ADA in lane11 of FIG. 4. The murine band in lanes 2-10 reveals that equal amountsof protein were loaded. These data demonstrate that transduction oflong-term reconstituting hematopoietic stem cells by supernatantinfection on FN 30/35 is equivalent to co-culture and far superior tosupernatant infection without FN 30/35.

EXAMPLE 8 Mechanism of Improved Transduction by Retrovirus VectorsBinding to FN 30/35

8.1. Experimental

To test whether increased transduction is the result of co-localizationof virus and hematopoietic cells, we analyzed whether recombinantretroviral particles demonstrate binding to FN 30/35. Therefore, FN30/35-coated plates were preincubated with supernatant containing TKNeovirus for 30 minutes and thereafter extensively washed. The viral titerof supernatant was determined using NIH/3T3 cells according to standardprocedures (Markowitz, D., S. Goff, and A. Bank, J. Virol., Vol. 62, pp1120-1124 (1988)). Briefly, 3T3 cells were plated at a concentration of1000 cells/well in a 6-well tissue culture plate and grown overnight.Serial dilutions of virus supernatant were added to each well with 7.5μ./ml polybrene and incubated for 2.5 hours at 37° C. after which 2 mlof medium was added. After 24 hours, the medium was replaced with mediumcontaining G418 (0.75 mg/ml, dry powder, Gibco) and the plates incubatedfor 10-12 days. The G418-resistant colonies (G418^(r) were stained after10-12 days and scored. The number of colonies/well multiplied by thedilution of virus supernatant was used as the infectious particles(cfu)/ml of supernatant. We assessed/“titered” the amount of retroviralparticles remaining on FN 30/35-coated or BSA-coated 35 mm plates afterpreincubation with virus supernatant and intensive washing by adding1000 NIH/3T3 per 35 mm bacteriologic dish cells plus polybrene.Twenty-four hours later, cultures were fed with medium containing 0.75mg/ml G418 (dry powder) and the cells further incubated for 10-12 days.Following this incubation, the presence of adherent virus wasquantitated by enumerating G418-resistant NIH/3T3 colonies.

To assess whether virus binding to FN 30/35 occurs in a dose-dependentfashion, the above experiments were repeated with increasingconcentrations of FN 30/35 coating the dishes. Therefore, 35 mmbacteriologic dishes were coated with 1, 4, 10 and 20 μg/cm² FN 30/35 asdescribed above. A 1:50 dilution of a TKNeo virus stock previouslytitered at 1×10⁴ infectious particles/ml was incubated on FN30/35-coated plates for 30 minutes. After intensive washing, 2000NIH/3T3 cells were added to each well. Selection was carried out asabove and G418-resistant NIH/3T3 colonies counted after 10 days ofselection.

8.2. Results

FIG. 5 sets forth the results of one of three representativeexperiments. Using TKNeo supernatant, retroviral titers measured byG41₈r colonies in NIH/3T3 cells were reduced by more than 3 logs (4×10³to 0) on BSA-coated plates, while titer reduction was only 1 log onplates coated with 30/35 FN. These data demonstrate that retrovirusquantitatively binds to FN 30/35 but does not bind to dishes coated withBSA (control dishes). FIG. 6 shows that increased numbers ofG418-resistant colonies were detected when virus-containing supernatantwas incubated on plates coated with increased concentrations of FN30/35. Therefore, virus binding to FN 30/35 occurs in a dose-dependentfashion.

EXAMPLE 9 Virus Binding to Recombinant Fibronectin Fragments

9.1. Experimental

Kimizuka et al. have previously reported the expression of cloned FN DNAsequences in E. coli (Kimizuka, F., Y. Taguchi, Y. Ohdate, Y. Kawase, T.Shimojo, D. Hashino, I. Kato, K. Sekiguchi, and K. Titani, J. Biochem.,Vol. 110, pp 284-291 (1991)). Cloned and chimeric peptides include oneor a combination of several important sequences in fibronectin known toparticipate in cell adhesion (including RGDS, CS-1 and heparin-bindingsite), see FIG. 7. To analyze whether retrovirus can bind to theserecombinant FN fragments, 3T3 cell colony formation assays were repeatedon plates coated with the recombinant fragments C-274, H-271, H-296,CH-271, CH-296, and CS-1 as well as FN 30/35 as a positive control,using two different dilutions (1:10 and 1:100) of the frozen retrovirusTKNeo stock with 1×10⁴ infectious particles/ml. FN fragments were usedat a concentration of 120-130 pmol/cm2 (equivalent to 4 μg/cm² forC-274, H-271, H-296, C-CS1, FN 30/35 and 8 μg/cm² for CH-271 andCH-296). Briefly, plates were coated, virus was added, plates wereextensively washed, NIH/3T3 cells were added for 24 hours and then grownin selection medium for 10 days, subsequently colonies were stained andcounted. 9.2. Results

FIG. 8 demonstrates that the number of the G418-resistant colonies (andtherefore virus adhesion) was increased in fragments H-271, H-296,CH-271 and CH-296. Furthermore, it shows that the amount of virus boundwas roughly comparable between these recombinant fragments and FN 30/35,although in this work the CH-271 fragment exhibited the highest level ofvirus binding. Common to all of these 5 FN fragments are the type IIIrepeats 12 14 which contain the high-affinity heparin-binding site(Ruoslahti, E., Ann. Rev. Biochem., Vol. 57, pp 375-413 (1988) andKimizuka, F., Y. Taguchi, Y. Ohdate, Y. Kawase, T. Shimojo, K. Hashino,I. Kato, K. Sekiguchi, and K. Titani, J. Biochem., Vol. 110, pp 284-291(1991)) probably located in repeat 13 (Kimizuka, F., Y. Taguchi, Y.Ohdate, Y. Kawase, T. Shimojo, K. Hashino, I. Kato, K. Sekiguchi, and K.Titani, J. Biochem., Vol. 110, pp 284-291 (1991)). This suggests thatvirus binding is occurring via this known adhesion site. This wasevidenced by pre-incubating dishes coated with 4 μg/cm² CH-271 withincreasing concentrations (10-1000 μg/ml) of heparin sulfate, a highlycharged molecule known to inhibit cell binding to the heparin-bindingsite. As seen in FIG. 9, the number of G418-resistant colonies isdecreased following pre-incubation of CH-271 with increasingconcentrations of heparin sulfate. These data suggest that virus bindingto FN is mediated through the high affinity heparin-binding site locatedimmediately adjacent to the CS-1 site in the carboxyl-terminal domainsof FN.

EXAMPLE 10 Transduction of Hematopoietic Cells on RecombinantFibronectin Fragments

10.1. Experimental

To analyze whether the increased transduction of hematopoietic cellsdescribed previously on FN 30/35 could also be seen with recombinant FNfragments, we assessed the transduction efficiency of supernatantinfections in vitro using high proliferative potential-colony formingcell (HPP-CFC) assays. By employing EAL2a vectors, we compared theinfluence of various recombinant FN fragments versus FN 30/35supernatant infection on BSA on transduction of hematopoietic cellsusing growth of G418-resistant colonies as an indicator of genetransfer. Furthermore, we compared the ability of virus particlesalready adherent to FN fragments (versus supernatant virus) to transducehematopoietic cells. 0.5 to 1×10⁶ prestimulated bone marrow cells wereincubated on 35 mm FN-coated petri dishes in 1-2 ml of EAL2a viruscontaining supernatant with growth factors and 5 mu.g/ml polybrene asdiscussed above. To assess transduction of hematopoietic cells by virusbound to FN fragments, 35 mm FN-coated dishes incubated withvirus-containing supernatant were washed three times with 2 ml PBS each.Subsequently, 0.5 to 1×10⁶ prestimulated bone barrow cells were added in2 ml of medium supplemented with growth factors and polybrene. 22 hourslater, cells were harvested and plated in HPP-CFC assays with andwithout 1.5 mg/ml G418 as described (Bradley, T. R. and D. Metcalf,Aust. J. Exp. Biol. Med. Sci., Vol. 44, pp 287-293 (1966)). The cultureswere incubated for 14 days in 7% CO₂ at 33° C. and the gene transferefficiency was calculated as the percentage of G418 resistant colonies.

10.2. Results

Transduction of primitive hematopoietic cells via supernatant infection(FIG. 10) was significantly higher than supernatant infection on BSA forall fragments which included the heparin-binding site (HBS) and at leastone more active cell adhesion site (solid bars). Transduction efficacyof the recombinant fragments CH-271, H-296, CH-296 and C-CS1 was similarto the effect of FN 30/35, all three fragments which include both theheparin-binding and the CS-1 site. In all other cases the transductionwas dramatically reduced. These data demonstrate that the increasedtransduction of primitive hematopoietic cells previously shown on FN30/35 can be replicated on recombinant FN fragments. It furtherdemonstrates that virus directly bound to fibronectin is capable ofgenetic transduction of hematopoietic cells. Finally it confirms theutility of the presence of both the CS-1 and the heparin binding sitefor transduction of primitive hematopoietic precursor (stem) cells.

EXAMPLE 11 Long-Term Bone Marrow Reconstitution of Mice UsingTransduction of Murine Donor Cells on Recombinant Fibronectin Fragments

11.1. Experimental

We repeated the above in vitro studies (from Example 10) for primitivehematopoietic progenitor cells comparing supernatant infection on BSA vsFN 30/35 versus recombinant FN fragments versus coculture using bonemarrow transplantation to analyze effects on reconstitutinghematopoietic stem cells. Briefly, lethally irradiated mice wereinjected with donor cells which were transduced with the EPHA-5 vectorscontaining the human ADA cDNA. After 1 month and after approximately 6months, gene transfer efficacy was analyzed from peripheral blood in ADAisoenzyme assays.

11.2. Results

FIG. 11 shows results after 1 month, and clearly shows the fibronectinfragment H-296 containing both the heparin binding site and the CS-1yields similar results to FN 30/35 and coculture. Fragments which do notcontain both these sites are less effective in transducing atransplantable hematopoietic cell. These data demonstrate thatco-localization of primitive hematopoietic/stem cells and retrovirusbound to recombinant FN fragments containing both the CS-1 and theheparin binding sites effectively transduces transplantablehematopoietic cells.

FIG. 12 shows results four and six months post-transplantation. At thistime point, genetic transduction of repopulating hematopoietic stemcells could not be demonstrated in animals transplanted with cellstransduced on control plates or on fragments containing only CBD (C-274)or ^(III)12-14 (H-271). Genetic transduction of HSCs was less frequentlyseen on the C-CS1 fragment, in this case ⅓ animals was positive for thehuman protein. Gene transfer in in vivo repopulating stem cells wasuniformly seen on fragments containing the HBD in combination witheither CBD (CH-271) or CS1 (H-296). Transduction on fragments containingall three cell binding sites (CH-296) was most efficient comparable withco-culture of target cells the producer cell line. These data show thatfibronectin fragments which contain ^(III) 12-14 in combination with thebinding site(s) for VLA-4 and/or VLA-5 are capable of increasingretroviral-mediated gene transfer into murine hematopoietic progenitorand stem cells.

EXAMPLE 12

Fibronectin directs cell adhesion through at least three sites (FIG.13): the cell binding domain (CBD) which contains the tetrapeptide RGDSin repeat 10 via the integrin VLA-5; the heparin binding domaincontained within the type III repeats 12-14 (^(III)12-14) via cellsurface proteoglycan molecules; and the CS1 sequence within thealternatively spliced IIICS region via the integrin VLA-4. In thesestudies, we utilized six chimeric recombinant FN fragments shown in FIG.13 which contain these single cell adhesion sites alone or incombinations in the peptide.

FN-coated bacterial dishes were incubated with 200 cfu in 2 ml ofsupernatent (SN) from the amphotropic packaging cell line TKNeo. After30 minutes at 37° C., dishes were washed three times with PBS and then2000 NIH/3T3 (fibroblast) cells were added in 2 ml of DMEM supplementedwith 10% calf serum (CS; Sigma, U.S.A.) and 2% P/S. The next day, cellswere put in selection medium with 0.75 mg/ml G418. 8-10 days later,dishes were stained with Stat Stain (Volu-Sol, U.S.A.) and G418.sup.rcolonies counted. By this assay, retrovirus does not bind to uncoated orBSA-coated plates. As shown in FIG. 14, gene transfer into NIH/3T3 cellsoccurred only on fragments which contained the Heparin-II bindingdomain, also referred to herein as “^(III)12-14”(H-271, CH-271, H-296,CH-296). These data demonstrate that retroviral particles directly bindto sequences within the ^(III)12-14 repeats of fibronectin. Infection ofNIH/3T3 cells was significantly higher on FN fragments containing both^(III)12-14 and CBD compared to ^(III)12-14 alone (compare H-271 versusCH-271). Remarkably, up to 80 G418^(r) NIH/3T3 colonies/plate weregenerated from an input of only 200 NEO cfu/plate. In contrast, additionof CS1 did not show any effect on NIH/3T3 transduction (H-271 versusH-296, CH-271 versus CH-296).

To evidence that the mechanism by which FN increased retroviral-mediatedgene transfer is by the simultaneous binding of retrovirus and targetcells to the fragment, experiments using a non-adherent cell line(HL60—a known promyelocytic leukemia cell line) were conducted. HL60cells were stained with directly fluorochrome-conjugated monoclonalantibodies against VLA-4 (FITC; Immunotech, U.S.A.) and VLA-5 (PE;Antigenix America, U.S.A.) or with the isotype controls for IgG1 andIgG2a (Becton Dickinson). Dead cells were excluded by propifium iodinestaining. Cells were then analyzed on a FACScan (Becton Dickinson). Forgene transfer studies, 104 HL60 cells were suspended with viralsupernatant from the amphotropic packaging cell line DAG (obtained fromSt. Jude's Children Research Hospital, Memphis, Tenn., U.S.A.)containing the extracellular domain of the nerve growth factor receptor(NGF-R) (titer 10⁵ cfu/ml) and then added to bacterial 6-well platescoated with the six FN fragments at a concentration of 2 μ/cm². After 4hours, 2 ml of conditioned medium from ongoing HL60 cultures was added.4 ml of fresh medium (RPMI (Gibco) with 5% FCS and 2% P/S) was addedafter 4-5 days. Cells were allowed to grow for a total of 8 days andthen stained with the monoclonal antibody 8211 against the NGF-R(Boehringer, U.S.A.) or with an isotype IgGl control (Dako, U.S.A.) andthen reacted with a secondary FITC-conjugated goat-anti-mouse serum(Boehringer). Samples were incubated with propidium iodine (PI) forexclusion of cell debris and subsequently measured on the FACScan. Genetransfer was demonstrated after gating for live cells by analysis of theNGF-R expression. All gene transfer studies were performed withoutPolybrene or protamine. As shown in FIG. 15, HL60 cells express VLA-4but not VLA-5 in flow cytometry analysis (A+B). Consistent withexpression data, HL60 cells adhered only to plates coated with fragmentscontaining CS1 (H-296, CH-296, C-CS1). As shown in FIG. 15, genetictransduction of HL-60 cells occurred only on fragments which contained^(III)12-14 in combination with CS1 (H-296, CH-296). Although HL-60cells adhere to the C-CS1 fragment via their VLA-4 integrin, the absenceof ^(III)12-14 to which retroviral binding occurs dramatically reducestransduction of HL60 cells.

In another set of experiments, increasing concentration of highmolecular weight (HMW) heparin (Sigma, U.S.A., MW about 7000 25000) weredissolved in 2 ml Hanks Balanced Salt Solution supplemented with 2.5%(v/v) 1 M Hepes (HBSS/Hepes; all Gibco) and added to bacterial 6-wellplates coated with the different FN fragments at a concentration of 4μg/cm². After 30 minutes plates were washed three times with PBS andthen 400 cfu/2 ml of the amphotropic TKNeo vector was added. After 30minutes at 37° C., plates were washed again with PBS and then 2000NIH/3T3 cells in DMEM with 10% CS and 2% P/S were added. Selection wasperformed as above and gene transfer efficiency was enumerated as thenumber of G418r colonies after 8-10 days of culture. In a similar set ofexperiments, fractionated low molecular weight (LMW) heparin (Sigma,U.S.A., MW about 3000) was dissolved in 2 ml of HBSS/Hepes at increasingconcentration. Experimental design was then as described above for HMWheparin. Gene transfer efficiency was read out as the number of G418rcolonies after 8-10 days. All of these gene transfer studies wereperformed without Polybrene or protamine. The results of theseexperiments are shown in FIG. 16. As shown, virus binding to FN can becompeted in a dose-dependent fashion by high (16A), but not lowmolecular weight heparin (16B). These results evidence that retroviralparticles bind to sequences within ^(III)12-14.

EXAMPLE 13 Selective Transduction of BFU-E Cells in CD34+ CellularPopulation

In this Example it was demonstrated that selected cells within a mixedcellular population can be targeted for transduction by using apolypeptide with a viral binding domain and a cell binding domain whichis specific for the targeted cells. The general TKNEO infection andassay protocols of Example 1 were repeated, except using a substantiallyhomogeneous population of CD34+ cells containing BFU-E, CFU-GM andCFU-Mix cells (obtained by affinity chromatography from cord blood (CB)cells), and using the recombinant FN fragment CH-271 at variedconcentrations of 1, 2, 4, 8, 16 and 20 μg/cm^(2 .) The results areshown in FIG. 17. As shown, BFU-E cells, which bind to the VLA-5 bindingsite, were transduced with high efficiencies (greater than 25% G418resistant colonies), whereas CFU-GM and CFU-Mix populations, which donot exhibit significant binding to VLA-5, were transduced atsubstantially lower efficiencies (less than about 5% G418 resistantcolonies).

EXAMPLE 14 Transduction of c-KIT+Cells

In this Example a cellular population substantially homogeneous as toc-KIT+ was transduced in accordance with the invention. Thus, c-KIT+cells were isolated from murine bone marrow using flow cytometricsorting techniques. The cells were subjected to an infection protocolusing the TKNEO vector generally as described in Example 1 exceptvarying the FN fragment as indicated in FIG. 18, and the infected cellswere assayed for HPP-CFC. As shown in FIG. 18, FN fragments whichcontained III.₁₂₋₁₄ and the VLA-4 binding site (FN 30/35, H296, andCH-296) led to high transduction efficiencies (greater than 80% G418resistant colonies) whereas FN fragments lacking these two domainsresulted in no significant transduction (C274 and C-CS1) orsubstantially lower efficiencies (H271, CH271, less than 20% G418resistant colonies).

EXAMPLE 15 Transduction of NIH/3T3 and Clonogenic BM Cells Using VaryingConcentrations of Polybrene

In this set of experiments it was demonstrated that advantageoustransduction methods of the invention are conducted in the absence ofhexadimethrine bromide. NIH/3T3 (fibroblast) cells and clonogenic bonemarrow cells were subjected to TKNEO infection protocols generally asdescribed in Examples 12 and 1, respectively, except that the vector,cells and varying concentrations of Polybrene were first suspendedtogether and then applied to a substrate coated with the FN fragmentCH-296 (8 μg/ml). Assays were as described previously for these celltypes. The results are given in FIGS. 19 and 20. As shown in FIG. 19,the number of G418 resistant NIH/3T3 colonies decreases dramaticallywith increasing Polybrene concentrations, ranging from about 14 when noPolybrene was used down to about 4 when 12.5 mu.g/ml Polybrene was used.Similarly, FIG. 20 reflects that nearly 40 colonies were observed whenthe protocol was conducted in the absence of Polybrene, whereas thecorresponding value when using 10 μg/ml was less than 15.

While the invention has been illustrated and described in detail in theforegoing description, the same is to be considered as illustrative andnot restrictive in character, it being understood that only thepreferred embodiment has been described and that all changes andmodifications that come within the spirit of the invention are desiredto be protected.

All publications cited herein are hereby incorporated by reference intheir entirety as if each had been individually incorporated byreference and fully set forth. In addition, co-pending U.S. patentapplication Ser. No. 08/218,355 filed Mar. 25, 1994, and co-pendingInternational Application Ser. No. PCT/US95/03817 filed Mar. 27, 1995and designating the United States, are hereby incorporated by referencein their entirety as is fully set forth.

1. A method for making a construct useful for enhancingretroviral-mediated DNA transfer into a predetermined target cell,comprising the steps of: selecting a ligand which binds with specificityto a predetermined target cell; and covalently coupling said ligand to apolypeptide containing an amino acid sequence which exhibits theretrovirus-binding activity of the Heparin-II domain of fibronectin. 2.The method for making a construct according to claim 1, wherein saidligand which binds with specificity to said predetermined target cell isselected from the group of ligands consisting of a cell adhesionproteins, cytokines, hormones, monoclonal antibodies, polyclonalantibodies, cell binding carbohydrates, cell metabolites, and functionalpolypeptides that bind cells.
 3. The method for making a constructaccording to claim 1, wherein said ligand which binds with specificityto said predetermined target cell is erythropoiten.
 4. The method formaking a construct according to claim 1, wherein said ligand which bindswith specificity to said predetermined target cell is G-CSF.
 5. Themethod for making a construct according to claim 1, wherein said ligandwhich binds with specificity to said predetermined target cell includesan amino acid sequence with at least 70 percent sequence identity to theCS-1 cell adhesion domain of fibronectin.
 6. The method for making aconstruct according to claim 1, wherein said ligand which binds withspecificity to said predetermined target cells includes an amino acidsequence with at least 90 percent sequence identity to the CS-1 celladhesion domain of fibronectin.
 7. The method for making a constructaccording to claim 1, wherein said ligand which binds with specificityto said predetermined target includes the CS-1 cell adhesion domain offibronectin (SEQ ID NO. 2) Asp Glu Leu Pro Gln Leu Val Thr Leu Pro HisPro Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr.


8. The method for making a construct according to claim 1, wherein saidligand which binds with specificity to said predetermined target cellhas at least 70 percent sequence identity to the CS-1 cell adhesiondomain of fibronectin.
 9. The method for making a construct according toclaim 1, wherein said ligand which binds with specificity to saidpredetermined target cell has at least 90 percent sequence identity tothe CS-1 cell adhesion domain of fibronectin.
 10. The method for makinga construct according to claim 1, wherein said ligand which binds withspecificity to said predetermined target is the amino acid sequence (SEQID NO. 2) Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu HisGly Pro Glu Ile Leu Asp Val Pro Ser Thr.


11. The method for making a construct according to claim 1, wherein saidpolypeptide which exhibits the retrovirus-binding activity of theHeparin-II domain of fibronectin includes an amino acid sequence havingleast at least 70 percent identity to the Heparin-II domain offibronectin.
 12. The method for making a construct according to claim 1,wherein said polypeptide which exhibits the retrovirus-binding activityof the Heparin-II domain of fibronectin includes an amino acid sequencehaving least at least 90 percent identity to the Heparin-II domain offibronectin.
 13. The method for making a construct according to claim 1,wherein said polypeptide which exhibits the retrovirus-binding activityof the Heparin-II domain of fibronectin includes the amino acid sequence(SEQ. I.D. NO. 1): Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr Gln ValThr Pro Thr Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn Val Gln Leu Thr GlyTyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro Met Lys Glu Ile AsnLeu Ala Pro Asp Ser Ser Ser Val Val Val Ser Gly Leu Met Val Ala Thr LysTyr Glu Val Ser Val Tyr Ala Leu Lys Asp Thr Leu Thr Ser Arg Pro Ala GlnGly Val Val Thr Thr Leu Glu Asn Val Ser Pro Pro Arg Arg Ala Arg Val ThrAsp Ala Thr Glu Thr Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr IleThr Gly Phe Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln ArgThr Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly ThrAsp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser Pro ValVal Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn Leu Arg Phe Leu AlaThr Thr Pro Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg Ile ThrGly Tyr Ile Ile Lys Tyr Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val ProArg Pro Arg Pro Gly Val Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly ThrGlu Tyr Thr Ile Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu Pro LeuIle Gly Arg Lys Lys Thr.


14. The method for making a construct according to claim 1, wherein saidpolypeptide which exhibits the retrovirus-binding activity of theHeparin-II domain of fibronectin has at least 70 percent identity to theamino acid sequence of the Heparin-II domain of fibronectin.
 15. Themethod for making a construct according to claim 1, wherein saidpolypeptide which exhibits the retrovirus-binding activity of theHeparin-II domain of fibronectin has at least 70 percent identity to theamino acid sequence of the Heparin-II domain of fibronectin.
 16. Themethod for making a construct according to claim 1, wherein saidHeparin-II domain of fibronectin is a polypeptide with an amino acidsequence having at least 90 percent identity to (SEQ. ID NO. 1): Ala IlePro Ala Pro Thr Asp Leu Lys Phe Thr Gln Val Thr Pro Thr Ser Leu Ser AlaGln Trp Thr Pro Pro Asn Val Gln Leu Thr Gly Tyr Arg Val Arg Val Thr ProLys Glu Lys Thr Gly Pro Met Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser SerVal Val Val Ser Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser Val Tyr AlaLeu Lys Asp Thr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu GluAsn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr IleThr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp AlaVal Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp Val ArgSer Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr Asp Tyr Lys Ile Tyr Leu TyrThr Leu Asn Asp Asn Ala Arg Ser Ser Pro Val Val Ile Asp Ala Ser Thr AlaIle Asp Ala Pro Ser Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu LeuVal Ser Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr GluLys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro Gly Val ThrGlu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Tyr Val IleAla Leu Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile Gly Arg Lys Lys Thr.